Production of microspheres

ABSTRACT

Protein microspheres are produced by contacting an aqueous solution of a macromolecule and a polymer with a surface at a high surface area to volume ratio, and heating the solution. The microspheres are useful for preparing pharmaceuticals of defined dimensions which can be delivered to a patient by inhalation therapy.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of PCTInternational application PCT/US01/51166, filed Oct. 25, 2001, which waspublished under PCT Article 21(2) in English, which claims priorityunder 35 U.S.C. §119 to U.S. Provisional Application Ser. No.60/244,098, filed Oct. 27, 2000, entitled “Production of Microspheres”,the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to a process for preparing protein microsphereshaving a high protein content. The protein microspheres of thisinvention are prepared by contacting an aqueous mixture of the proteinand a polymer with a surface having a high ratio of surface area tovolume. The protein microspheres are suitable for preparingpharmaceutical compositions which can be delivered to a patient,principally by pulmonary, parenteral and oral administration routes. Theprocess can be operated as a continuous process for increased efficiencyand productivity.

The preparation and delivery of therapeutic proteins of interest is anarea of concentrated research and development activity in thepharmaceutical industry. It is highly desirable to formulate proteinswith select release characteristics in the patient with maximum clinicaleffectiveness. For pulmonary administration, the protein is ideallyprepared in the form of discrete microspheres, which are solid orsemi-solid particles having a diameter of between 0.5 and 5.0 microns.It is also desirable for the particles to have a protein content as highas possible for maximum therapeutic effectiveness.

Microspheres have been commercially available for biochemical andbiotherapeutic applications for many years. For example, antibodiesconjugated to beads produce relatively large particles which arespecific for a particular ligand. These large antibody-coated particlesare used to bind receptors on the surface of a cell for cellularactivation, for binding to a solid phase for immunoaffinitypurification, and for the delivery of therapeutic agents to a targetusing tissue or tumor-specific antibodies. The beads can be formed fromsynthetic polymers or proteins, although synthetic polymers aresometimes preferred due to durability and cost.

Microparticles produced by standard production methods frequently have awide particle size distribution, lack uniformity, fail to provideadequate release kinetics, and are difficult and expensive to produce.Furthermore, the polymers used to prepare these microspheres are usuallysoluble in organic solvents, requiring the use of special facilitiesdesigned to handle organic solvents. The organic solvents can denatureproteins or peptides contained in the microspheres, and may also betoxic when administered to humans or animals.

In addition, the microparticles may be large and tend to formaggregates, requiring a size selection process to remove particlesconsidered to be too large for administration to patients by injectionor inhalation. This requires sieving and resulting product loss. Largesize particles can also require the use of large gauge needles forinjection, often causing discomfort for the patient.

Currently available microspheres are designed to release proteins in anaqueous medium, by incorporating the proteins into a hydrophobicerodible or non-erodible matrix. Many particles exhibit release kineticsbased on both erosion and diffusion. In this type of system, an initialburst or rapid release of the drug is observed. This burst effect oftenresults in unwanted side effects in some patients.

U.S. Pat. No. 5,981,719, U.S. Pat. No. 5,849,884 and U.S. Pat. No.6,090,925, the disclosures of which are incorporated by reference hereinin their entirety, describe microparticles formed by combining amacromolecule, such as a protein or peptide, and a polymer in an aqueoussolution at a pH near the isolelectric point of the macromolecule. Thesolution is heated to prepare microparticles having a protein content ofgreater than 40%. The microparticles thus formed comprise a matrix ofsubstantially homogeneous, intertwined macromolecules and polymers,which permit the aqueous medium to enter and solubilize the componentsof the microparticle. The microparticles can be designed to exhibitshort-term or long-term release kinetics, providing either rapid orsustained release characteristics.

U.S. Pat. No. 6,051,256 relates to processes for preparing powders ofbiological macromolecules by atomizing liquid solutions of themacromolecules, drying the droplets, and collecting the resultingparticles. Biological macromolecules which can be used in this processinclude insulin and calcitonin.

It will be appreciated that there is a continuing need for a process forpreparing and delivering biological agents as microspheres to maximizetheir effectiveness and minimize the safety concerns for the therapeuticagent.

SUMMARY OF THE INVENTION

The invention provides a process for the production of microspheres ofbiological molecules of interest. The microspheres are useful astherapeutic or diagnostic agents for treating or diagnosing diseasestates in a subject in vivo or in vitro. The microspheres areparticularly useful as active therapeutic components of inhalers forpulmonary administration to human patients.

The process comprises combining the macromolecule and a polymer in anaqueous solution, contacting a volume of the solution with a surfacehaving a surface area to volume ratio of at least about 6.5 cm⁻¹,preferably at least about 14 cm⁻¹, and exposing the solution to anenergy source for a sufficient period of time to form microspheres.Preferably, the surface is a hydrophobic surface, such as a hydrophobicpolymer, or a metal or ceramic material. Particularly preferred surfacesinclude stainless steel, polypropylene, polystyrene, PTFE and siliconepolymers. The microspheres form as a result of the interaction of thesolution with the surface, which functions as a site of nucleation forthe formation of microspheres.

In one aspect of the invention, the microspheres are substantiallyspherical in shape, and have mean diameters within the range of fromabout 0.1 microns and 10.0 microns. Preferably, the mean diameter of themicrospheres is within the range of from about 0.5 microns and 5.0microns, and more preferably within a range of from about 1.0 micronsand 2.0 microns.

The macromolecules which are useful in the practice of this inventioninclude both therapeutic and diagnostic agents. Therapeutic agentsinclude antibiotics, hematopoietics, antiinfective agents, antiulceragents, antiallergic agents, antipyretics, analgesics, antiinflammatoryagents, antidementia agents, antiviral agents, antitumoral agents,antidepressants, psychotropic agents, cardiotonics, antiarrythmicagents, vasodilators, antihypertensive agents, antidiabetic agents,anticoagulants, and cholesterol lowering agents. Other examples ofsuitable macromolecules include proteins, peptides, nucleic acids,carbohydrates, protein conjugates, virus, virus particles, and mixturesthereof. These macromolecules are characterized by the ability tointeract with the polymer in the presence of an energy source, such asheat, to form intact, discrete microspheres having a high content ofmacromolecule. Preferably, the macromolecule comprises at least about90% by weight of the microspheres, more preferably at least about 95% byweight, and most preferably at least about 99%. In especially preferredembodiments, the microsphere has a sufficiently high protein content tobe indistinguishable from the protein standard.

Preferred macromolecules include peptides, such as polypeptides,carbohydrates, such as polysaccharides, proteins, and particularlytherapeutic proteins such as insulin, human serum albumin, human growthhormone, parathyroid hormone and calcitonin.

In another aspect, the energy source used to form the microspheres isheat energy. Heat can be applied to the aqueous solution containing thecomponents for the production of the microspheres to heat the solutionto a temperature in the range of from about 37° C. to about 95° C. for atime period of about 1 minute to about 24 hours. The aqueous solutioncan contain water, water-miscible or water soluble solvents, such asethanol, pyrrolidone, 2-pyrrolidone, DMSO, acetone and the like.

In a further aspect, the polymer incorporated in the aqueous solution ispreferably selected from the group consisting of carbohydrate polymers,polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids,polyorganic acids, polyamino acids, polyethers, naturally occurringpolymers, polyimids, polyesters, polyaldehydes, co-polymers, blockco-polymers, terpolymers, surfactants, branched polymers,cyclo-polymers, and mixtures thereof. More preferably, the polymer isdextran, polyethylene glycol, polyvinyl pyrrolidone, co-polymers ofpolyethylene glycol and polyvinyl pyrrolidone, polyvinyl alcohol, orco-polymers of polyoxyethylene and polyoxypropylene, and mixturesthereof. Most preferably, the polymer is a co-polymer of polyethyleneglycol and polyvinyl pyrrolidone, or a co-polymer of polyoxyethylene andpolyoxypropylene.

The polymer is a water soluble polymer which is capable of removingwater from the macromolecule, or dehydrating the macromolecule. Suitablepolymers include, in addition to the specific polymers mentioned above,high molecular weight linear or branched chain polymers which aresoluble in water, or in a water miscible solvent, or both. Typical watersoluble polymers which are useful in this invention are described inpending, commonly assigned U.S. patent application Ser. No. 09/420,361,filed Oct. 18, 1999, the disclosure of which is incorporated herein inits entirety.

In a still further aspect, an apparatus which can be used to prepare themicrospheres of the present invention comprises a reactor having aninternal surface to volume ratio of 6.5 cm⁻¹, and means for heating theaqueous solution. The reactor contains an aqueous solution of amacromolecule and a polymer. The internal surface contacts the aqueoussolution and increases the internal surface area of the reactor.Preferably, the internal surface is provided by a bed of inert, solidmaterial, such as a bed of pellets, balls, plates, and the like,Alternatively, the internal surfaces can include tubes through which thesolution passes.

In order to prepare the microspheres of the present invention, thesurface area of the internal surface should be sufficient to provide asurface area to volume ratio of at least about 6.5 cm⁻¹, and preferablyat least about 14 cm⁻¹. Preferably, the surfaces are formed from ahydrophobic material, particularly hydrophobic polymers such aspolypropylene, polystyrene, PTFE or silicone polymers. Alternatively,the surfaces can be formed from metals, ceramics or glass. A preferredmetal is stainless steel. The surface functions as a site of nucleationfor the formation of microspheres.

The apparatus for preparing the microspheres of this invention can beoperated in a batch or continuous mode. Continuous mode operation istypically more efficient and cost effective than batch mode.

In yet another aspect, the invention includes devices and methods fordelivering the microspheres to a subject, and particularly a humanpatient in need of medical treatment. Suitable delivery routes includeparenteral, such as i.m., i.v. and s.c., and non-parenteral, such asoral, buccal, intrathecal, nasal, pulmonary, transdermal, transmucosal,and the like. Delivery devices include syringes, both needleless andneedle containing, and inhalers.

The syringe can contain a single dose of the microspheres for treating acondition that is treatable by the sustained release of themacromolecule in vivo. The number of microspheres present in the singledose is dependent on the type and activity of the macromolecule.Preferably, the single dose is selected to achieve sustained releaseover a period of time which has been optimized for treating theparticular medical condition.

An inhaler device can be utilized when it is desired to deliver atherapeutic dose of protein microspheres to the lung of a subject. Theprotein microspheres are prepared by contacting an aqueous solution ofthe protein and a polymer with a surface, and heating the solution toform the microspheres. The protein is preferably a therapeutic protein,such as insulin, human growth hormone, calcitonin or parathyroidhormone, and the protein content of the microspheres is preferably atleast about 90% or more, more preferably 95% or more, and mostpreferably at least about 99% or more. For pulmonary administration, themicrospheres are ideally sized to have a mean diameter in the range offrom about 0.5 microns to 5.0 microns, and preferably between 1 and 2microns.

The inhaler can be used to treat any medical condition in which theprotein can be administered by inhalation therapy. Typical inhalerdevices include dry powder inhalers, metered dose inhalers, nebulizersand electrostatic delivery devices. Typical applications of the deliveryapparatus of this invention include the deep lung delivery of insulinand similar proteins.

The microspheres of this invention can be prepared without the need forspray drying or milling processes. These microspheres are homogenous insize and shape, and reproducible between different batches. The proteinmicrospheres have been found to be unexpectedly stable and to formsuperior suspensions in the presence of propellants, both freon-basedand freon substitutes, that are commonly used in inhalers for pulmonarydelivery. It is believed that this increased stability may be due to thefact that the microspheres of this invention are dehydrated as a resultof the interaction of the polymer and the macromolecule, and haveincreased stability in the presence of inhaler propellants.

Although pulmonary delivery is preferred, the microspheres of thisinvention can be delivered orally, intranasally, intravenouslyintramuscularly, subcutaneously, and by other delivery methods suitablefor the delivery of therapeutic molecules.

These and other aspects of the invention will be described in greaterdetail herein. All technical and scientific terms have the meaningascribed herein, or if not so ascribed, as commonly understood by one ofordinary skill in this art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the number of particles, surface area averageand volume average for microspheres of the invention as a function ofthe diameter of the particles. The microspheres depicted in the figureare prepared from polyethylene glycol, polyvinyl pyrrolidone andinsulin.

FIG. 2 is a scanning electron micrograph (SEM) of insulin aggregatesprepared by a procedure other than the process of this invention. Anamorphous mass of lyophilized insulin is shown.

FIG. 3 is a scanning electron micrograph of 1 to 2 micron insulinmicrospheres prepared using polypropylene shards according to theprocess of this invention.

FIG. 4 is a scanning electron micrograph showing insulin aggregatesprepared without the use of the polypropylene shards of this invention.As shown in the micrograph, the vast majority of the mass constitutesaggregated material with very few actual microspheres.

FIGS. 5A, 5B and 5C are graphical representations showing thedifferential volume, differential number and differential surface area,respectively, of microspheres prepared without the use of polypropyleneshards, as a function of the particle diameter.

FIG. 6 is a scanning electron micrograph of insulin microspheres formedby a continuous flow process utilizing the method of this invention.

FIG. 7 is a graph showing the depression of blood glucose levels inrats. The blood glucose levels are plotted against the time afterinsulin injection for insulin microspheres and solubilized insulinmicrospheres. A saline control is also shown.

DETAILED DESCRIPTION OF THE INVENTION

A process for preparing microspheres having therapeutic and diagnosticapplications is described herein. The process involves combining amacromolecule and a polymer in an aqueous solution, and exposing theaqueous solution to an energy source. According to the process of thisinvention, a volume of the aqueous solution contacts a surface underconditions wherein the surface area to volume ratio of at least about6.5 cm⁻¹, and preferably at least about 14 cm⁻¹. The surface serves as asite of nucleation promoting the formation of the microspheres.

Therapeutic and diagnostic applications of the microspheres include drugdelivery, vaccination, gene therapy, and in vivo tissue or tumorimaging. Routes of administration include oral or parenteraladministration; mucosal administration; ophthalmic administration;intravenous, subcutaneous, intra articular, or intramuscular injection;inhalation administration; and topical administration.

The term “microspheres”, as used herein, denotes particles substantiallyspherical in shape having dimensions generally of between about 0.1microns and 10.0 microns in diameter. The microspheres typically exhibita narrow size distribution, and are formed as discrete particles.

An “aqueous solution”, as that term is used herein, includes solutionsof water alone, or water mixed with one or more water-miscible solvents,such as ethanol, DMSO, acetone and methyl pyrrolidone, 2-pyrrolidone.

The microspheres are produced by mixing macromolecules in an aqueousmixture with a water soluble polymer or mixture of polymers, thereaftercontacting the solution with an energy source, preferably heat, underconditions sufficient to form the microspheres. The solution ispreferably an aqueous solution. Either the macromolecule solution isadded to the polymer, or the polymer solution is added to themacromolecule solution, to cause removal of water from, or dehydrationof, the macromolecule. This process is also referred to by those skilledin the art as volume exclusion.

The macromolecule and polymer solution is then exposed to an energysource, such as heat, radiation, including microwave radiation, orionization, alone or in combination with sonication, vortexing, mixingor stirring, for a predetermined length of time to form and stabilizethe microspheres. The resulting microspheres are then separated from anyunincorporated components present in the solution by physical separationmethods well known to those skilled in the art, and may then be washedor exposed to other drug-containing solutions for binding of additionaldrugs to the microspheres.

The length of incubation time is dependent upon the respectiveconcentrations of polymer and macromolecule and the level of energy ofthe energy source. Microsphere stabilization can begin to occurimmediately upon exposure to the energy source. Preferably, themacromolecule and polymer mixture is heated at a temperature greaterthan room temperature for between approximately 1 minute and 24 hours.Most preferably, the polymer and macromolecules are heated for 30minutes or less at a temperature between approximately 37° C. and 95° C.

The formation of the microspheres according to this invention requires asite of nulceation which is commonly the vessel or container for themacromolecule and polymer aqueous solution. This is accomplished bycombining the macromolecule and polymer under conditions sufficient toprovide a surface area to volume ratio of at least about 6.5 cm⁻¹, andpreferably at least about 14 cm⁻¹. The surfaces can be formed from ahydrophobic material, such as a hydrophobic polymer, such aspolypropylene. Alternatively, the surface can be formed from a metal, aceramic or glass.

The vessel may inherently have a high surface area to volume ratio, asin the case of a tubular reactor, so that no adjustment with respect tothis ratio is required. Alternatively, the surface area to volume ratioin a vessel can be increased by adding materials to the vessel to reducethe internal volume relative to the surface area. This can beaccomplished by using a bed of particles, plates, or beads, for example,in the reactor.

The macromolecule component of the microsphere is any molecule having atertiary and quaternary structure or capable of having a tertiary andquaternary structure. Most preferably, the macromolecule is abiomolecule such as a protein, including enzymes and recombinantproteins, peptides, carbohydrates, polysaccharides, carbohydrate- orpolysaccharide-protein conjugates, nucleic acids, virus, virusparticles, conjugates of small molecules (such as a hapten) andproteins, or mixtures thereof. An organic or inorganic natural orsynthetic pharmaceutical compound or drug may be incorporated into themicrospheres by attaching the drug to a macromolecule, such as aprotein, and then forming the microspheres from the macromolecule-drugcomplex or conjugate. It will be understood by those skilled in the artthat a compound incapable of having a tertiary and quaternary structurecan be formed into a microsphere by incorporation or coupling of thecompound into a carrier molecule that has a tertiary and quaternarystructure. It will be further understood by those skilled in the artthat the macromolecule can be a portion of a molecule such as, forexample, a peptide, a single-stranded segment of a double-strandednucleic acid molecule, or a virus particle, having a tertiary andquaternary structure. It will also be understood that the term“macromolecule” includes a plurality of macromolecules and includescombinations of different macromolecules such as a combination of apharmaceutical compound and an affinity molecule for targeting thepharmaceutical compound to a tissue, organ or tumor requiring treatment.It will be further understood that an affinity molecule can be eitherthe receptor portion or the ligand portion of a receptor-ligandinteraction. Examples of ligands that interact with other biomoleculesinclude viruses, bacteria, polysaccharides, or toxins that act asantigens to generate an immune response when administered to an animaland cause the production of antibodies.

Suitable compounds or macromolecules include, but are not limited to,betaxolol™, diclofenac™, doxorubicin, rifampin™, leuprolide acetate,luteinizing hormone releasing hormone (LHRH), (D-Tryp6)-LHRH, nafarelinacetate, insulin, sodium insulin, zinc insulin, protamine, lysozyme,alpha-lactalbumin, basic fibroblast growth factor (bFGF),beta-lactoglobulin, trypsin, calcitonin, parathyroid hormone, carbonicanhydrase, ovalbumin, bovine serum albumin (BSA), human serum albumin(HSA), phosphorylase b, alkaline phosphatase, beta-galactosidase, IgG,fibrinogen, poly-L-lysine, IgM, DNA, desmopressin acetate, growthhormone releasing factor (GHRF), somatostatin, antide, Factor VIII,G-CSF/GM-CSF, human growth hormone (hGH), beta interferon, antithrombinIII, alpha interferon, alpha interferon 2 b.

The incubation conditions are typically optimized to incorporate atleast about 90%, preferably at least about 95%, and most preferably atleast about 99%, of the macromolecule in the reaction mixture byadjusting the pH, temperature, concentration of macromolecule, orduration of reaction or incubation. In general, less energy is requiredto form microspheres at higher concentrations of macromolecule.

Microspheres composed of nucleic acids are preferably prepared by firstmixing the nucleic acid either with a protein, such as bovine serumalbumin, or, because nucleic acids are anions, by the addition of acation, such as polylysine, which aids greatly in the formation ofmicrospheres.

As mentioned above, a small molecule or compound incapable of having atertiary and quaternary structure, such as a peptide or pharmaceuticalcompound, can be formed into a microsphere by incorporation or couplingof the compound into a carrier molecule that has a tertiary andquaternary structure. This may be achieved in several ways. For example,microspheres may be formed as described herein using a macromoleculehaving a tertiary and quaternary structure, such as a protein, and thenthe small molecule or compound is bound inside and/or on the surface ofthe microsphere. Alternatively, the small molecule or compound is boundto the macromolecule having a tertiary and quaternary structure usinghydrophobic or ionic interactions, and then microspheres are formed fromthe macromolecule-small molecule complex using the method describedherein. A third way to make microspheres from small molecules is toprepare microspheres using a macromolecule having a tertiary andquaternary structure in such a way that the microsphere has a net chargeand then add a small molecule or compound having an opposite net chargeso that the small molecule is physically attracted to and remainsattached to the microsphere, but can be released over time under theappropriate conditions. Alternatively, different types of non-covalentinteractions such as hydrophobic or affinity interactions may be used toallow attachment and subsequent release of small molecules.

When preparing microspheres containing a protein, a protein stabilizersuch as glycerol, fatty acids, sugars such as sucrose, ions such aszinc, sodium chloride, or any other protein stabilizers known to thoseskilled in the art may be added prior to the addition of the polymersduring microsphere formation to minimize protein denaturation.

Molecules, distinct from the macromolecules of which the microspheresare composed, may be attached to the outer surface of the microspheresby methods known to those skilled in the art to “coat” or “decorate” themicrospheres. The ability to attach molecules to the outer surface ofthe microsphere is due to the high concentration of macromolecule in themicrosphere. These molecules are attached for purposes such as tofacilitate targeting, enhance receptor mediation, and provide escapefrom endocytosis or destruction. For example, biomolecules such asphospholipids may be attached to the surface of the microsphere toprevent endocytosis by endosomes; receptors, antibodies or hormones maybe attached to the surface to promote or facilitate targeting of themicrosphere to the desired organ, tissue or cells of the body; andpolysaccharides, such as glucans, or other polymers, such as polyvinylpyrrolidone and PEG, may be attached to the outer surface of themicrosphere to enhance or to avoid uptake by macrophages.

In addition, one or more cleavable, erodilbe or soluble molecules may beattached to the outer surface of or within the microspheres. Thecleavable molecules are designed so that the microspheres are firsttargeted to a predetermined site under appropriate biological conditionsand then, upon exposure to a change in the biological conditions, suchas a pH change, the molecules are cleaved causing release of themicrosphere from the target site. In this way, microspheres are attachedto or talen up by cells due to the presence of the molecules attached tothe surface of the microspheres. When the molecule is cleaved, themicrospheres remain in the desired location, such as within thecytoplasm or nucleus of a cell, and are free to release themacromolecules of which the microspheres are composed. This isparticularly useful for drug delivery, wherein the microspheres containa drug that is targeted to a specific site requiring treatment, and thedrug can be slowly released at that site. The preferred site of cleavageis a diester bond.

The microspheres may also be coated with one or more stabilizingsubstances, which may be particularly useful for long term depoting withparenteral administration or for oral delivery by allowing passage ofthe microspheres through the stomach or gut without dissolution. Forexample, microspheres intended for oral delivery may be stabilized witha coating of a substance such as mucin, a secretion containingmucopolysaccharides produced by the goblet cells of the intestine, thesubmaxillary glands, and other mucous glandular cells.

Additionally, the microspheres can be non-covalently coated withcompounds such as fatty acids or lipids. The coating may be applied tothe microspheres by immersion in the solubilized coating substance,spraying the microspheres with the substance or other methods well knownto those skilled in the art.

In certain of the preferred embodiments, the microspheres of theinvention include a protein and at least one water soluble polymer. Asdiscussed above, the microspheres are formed by contacting the proteinand at least one water soluble polymer under aqueous conditions, and themicrospheres are then formed and stabilized by exposing the microspheresto an energy source, preferably heat, under conditions (e.g.,concentration, temperature) which result in microspheres which areresistant to physical and chemical treatments such as sonication andcaustic solutions.

In general, the microspheres of the invention are formed by mixing theprotein together with at least one water soluble polymer under suitableconditions which, preferably, permit the water soluble polymer to removewater from (“dehydrate”) the protein within specified or preferredratios (wt/wt) of protein to water soluble polymer (e.g., ratios rangefrom about 1 protein: 1 polymer to about 1 protein: 1000 polymer). Thepreferred ratio of protein to water soluble polymer in the microsphereformation reaction is in the range from about 1 protein: 5 polymer toabout 1 protein: 30 polymer. As noted above, a “water soluble polymer”of the invention refers to a polymer or mixture of polymers which,preferably, are capable of interacting with the macromolecule (e.g.,protein or other molecule) to cause volume exclusion.

Suitable water soluble polymers include soluble linear or branchedpolymers, preferably those having a high molecular weight. Polymers canbe highly water soluble, moderately-water soluble, or slightly watersoluble (greater than 2% wt/vol water soluble). The preferred watersoluble polymers are water soluble or soluble in a water misciblesolvent. The water soluble polymers may be solubilized by first beingdissolved in water, an aqueous buffered solution, or a water misciblesolvent and then combining the polymer solution with an aqueous solvent.In one embodiment, the water soluble polymer is a carbohydrate-basedpolymer. The preferred polymer is polyvinylpyrrolidone, polyethyleneglycol, dextran, polyoxyethylene-polyoxypropylene copolymer, polyvinylalcohol, starch, hetastarch, or mixtures thereof, the characteristics ofwhich are described in more detail below. The polymer or polymer mixturemay be prepared in accordance with the methods set forth in U.S. Pat.No. 5,525,519 to James E. Woiszwillo, or PCT Patent Application No.US93-00073 (International Publication No. WO 93/14110), filed Jan. 7,1993 and published on Jul. 22, 1993 by James E. Woiszwillo, both ofwhich are incorporated herein by reference), in which the polymer isdissolved in water or an aqueous solution, such as a buffer, in aconcentration between approximately 1 and 50 g/100 ml depending on themolecular weight of the polymer. The preferred total polymerconcentration in the polymer solution is between 10% and 80%, expressedas weight/volume percent. The preferred concentration of each polymer inthe polymer solution is between 5% and 50%.

Polyoxyethylene-polyoxypropylene copolymer, also known as poloxamer, issold by BASF (Parsippany, N.J.) and is available in a variety of formswith different relative percentages of polyoxyethylene andpolyoxypropylene within the copolymer.

PVP is a non-ionogenic, hydrophilic polymer having a mean molecularweight ranging from approximately 10,000 to 700,000 and the chemicalformula (C₆H₉NO)[n]. PVP is also known aspoly[1-(2-oxo-1-pyrrolidinyl)ethylene], Povidone™, Polyvidone™, RP 143™,Kollidon™, Peregal ST™, Periston™, Plasdone™, Plasmosan™, Protagent™,Subtosan™, and Vinisil™. PVP is non-toxic, highly hygroscopic andreadily dissolves in water or organic solvents.

Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is acondensation polymer of ethylene oxide and water having the generalchemical formula HO(CH₂CH₂O)[n]H.

Dextran is a term applied to polysaccharides produced by bacteriagrowing on a sucrose substrate. Native dextrans produced by bacteriasuch as Leuconostoc mesenteroides and Lactobacteria dextranicum usuallyhave a high molecular weight. Dextrans are routinely available and areused in injectable form as plasma expanders in humans.

Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates byreplacement of the acetate groups with hydroxyl groups and has theformula (CH₂CHOH)[n]. Most polyvinyl alcohols are soluble in water.

PEG, dextran, PVA and PVP are commercially available from chemicalsuppliers such as the Sigma Chemical Company (St. Louis, Mo.).

Preferably, the polymer is a polymer mixture containing an aqueoussolution of PVP having a molecular weight between 10,000 and 360,000,most preferably 40,000, and PEG having a molecular weight between 200and 35,000. PVP having a molecular weight of 40,000 and PEG having amolecular weight of 3500 is preferred. Preferably, the PVP is dissolvedin an acetate buffer and PEG is added to the aqueous PVP solution. Theconcentration of each polymer is preferably between 1 and 40 g/100 mldepending of the molecular weight of each polymer. Equal concentrationsof PVP and PEG generally provide the most favorable polymer mixture forthe formation of microspheres.

An alternative preferred polymer is a dextran, having a molecular weightfrom approximately 3000 to 500,000 daltons.

The volume of polymer added to the macromolecule varies depending on thesize, quantity and concentration of the macromolecule. Preferably, twovolumes of the polymer mixture at a 5-50% total polymer concentrationare added to one volume of a solution containing the macromolecule,typically at a concentration of 10 mg/ml. The polymer is present in aliquid phase during the reaction with macromolecule.

Applicants have discovered that contacting a volume of the aqueoussolution with a surface having a surface area to volume ratio of atleast about 6.5 cm⁻¹, preferably at least about 14 cm⁻¹, results in theformation spherically shaped microspheres rather than aggregates andother amorphous forms of particles. Preferably, the surface is ahydrophobic surface, such as a hydrophobic polymer, or a metal orceramic material. Particularly preferred surfaces include stainlesssteel, polypropylene, polystyrene, PTFE and silicone polymers. Thesurfaces can take the form of tubes, or a bed of pellets, balls, plates,etc. The microspheres are believed to form as a result of theinteraction of the solution with the surface, which functions as a siteof nucleation for the formation of microspheres.

The process can be operated in a batch or continuous mode. Continuousmode operation is typically more efficient and cost effective than batchmode.

The preferred energy source is heat. However, it will be understood bythose skilled in the art that other energy sources include heat,radiation, and ionization, alone or in combination with sonication,vortexing, mixing or stirring. Microsphere formation can occurimmediately upon exposure to the energy source or may require anextended exposure to the energy source depending on the characteristicsof the components and conditions. Preferably, the macromolecule-polymersolution mixture, is incubated in a water bath at a temperature greaterthan or equal to 37° C. and less than or equal to 95° C. for betweenapproximately 1 minute and 24 hours. Most preferably, the mixture isincubated for 5-30 minutes at a temperature between 50° C. and 90° C.The maximum incubation temperature is determined by the characteristicsof the macromolecule and the ultimate function of the microsphere.

The formed microspheres are separated from the non-incorporatedcomponents of the incubation mixture by conventional separation methodswell known to those skilled in the art. Preferably, the incubationmixture is centrifuged so that the microspheres sediment settles to thebottom of the centrifuge tube and the non-incorporated components remainin the supernatant, which is then removed by decanting. Alternatively, asuspension containing formed microspheres is filtered so that themicrospheres are retained on the filter and the non-incorporatedcomponents pass through the filter.

Further purification of the microspheres is achieved by washing in anappropriate volume of a washing solution. The preferred washing solutionwater, or a water-miscible solvent capable of removing the water solublepolymers. Repeated washings can be utilized as necessary and themicrospheres separated from the wash solution as described above.

As mentioned above, the characteristics of the microspheres can bealtered by manipulating the incubation conditions. For example, therelease kinetics of the microspheres may be retarded by increasing thereaction temperature or extending the length of reaction time duringmicrosphere formation. Release kinetics are also manipulated by choosingdifferent polymers, different concentrations of polymers, or differentratios of polymers used in the formation of the microspheres.

Microsphere size, shape and release kinetics can also be controlled byadjusting the microsphere formation conditions. For example, microsphereformation conditions can be optimized to produce smaller or largermicrosplieres, or the overall incubation time or incubation temperaturecan be increased, resulting in microspheres which have prolonged releasekinetics.

According to yet another aspect of the invention, a pharmaceuticalcomposition of matter and method for producing same are provided. Thecomposition includes a container containing a single dose ofmicrospheres containing an active agent for treating a condition that istreatable by the sustained release of an active agent from themicrospheres. The number of microspheres in the single dose is dependentupon the amount of active agent present in each microsphere and theperiod of time over which sustained release is desired. Preferably, thesingle dose is selected to achieve the sustained release of the activeagent over a period of about 1 to about 180 days with the desiredrelease profile.

According to another aspect of the invention, a syringe-containingcomposition is provided. The composition includes a syringe containing asingle dose of microspheres containing an active agent for treating acondition that is treatable by the sustained release of the active agentfrom the microspheres; and a needle attached to the syringe, wherein theneedle has a bore size that is from 14 to 30 gauge.

The preferred microspheres of the invention can also be prepared to havedimensions which permit the delivery of microspheres using a needlelesssyringe, thereby eliminating the disposal problems inherent with needleswhich must be disposed as a biohazard waste product. Thus, according toa particularly preferred aspect of the invention, a needleless syringecontaining one or more doses of microspheres containing an active agentfor treating a condition is provided.

The microspheres can also be prepared to have qualities suitable to bedelivered by other parenteral and non-parenteral routes such as oral,buccal, intrathecal, nasal, pulmonary, transdermal, transniucosal andthe like.

An inhaler device can be utilized for pulmonary delivery of atherapeutic dose of protein microspheres to the lung of a subject. Forpulmonary administration, the microspheres are ideally sized to have amean diameter in the range of from about 0.5 microns to 5.0 microns, andpreferably between 1 and 2 microns.

The inhaler can be used to treat any medical condition in which theprotein can be administered by inhalation therapy. Typical inhalerdevices include dry powder inhalers, metered dose inhalers, nebulizersand electrostatic delivery devices. Typical applications of the deliverydevice includes the deep lung delivery of insulin and similar proteins.

The protein microspheres have been found to be unexpectedly stable inthe presence of propellants, both freon-based and freon substitutes,commonly used in inhalers for pulmonary delivery. Without wishing to bebound by any theory or mechanism of operability, it is believed thatthis increased stability may be due to the fact that the microspheres ofthis invention are dehydrated as a result of the interaction of thepolymer and the macromolecule. This allows the microspheres to remainintact for prolonged periods of time in the presence of inhalerpropellants.

When used therapeutically, the microspheres of the invention areadministered in therapeutically effective amounts. In general, atherapeutically effective amount means that amount of macromoleculenecessary to affect a therapeutic response or to delay the onset of,inhibit the progression of, or halt altogether the particular conditionbeing treated. Generally, a therapeutically effective amount will varywith the subject's age, condition, and sex, as well as the nature andextent of the disease in the subject, all of which can be determined byone of ordinary skill in the art. The dosage may be adjusted by theindividual physician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount of active agenttypically varies from 0.01 mg/kg to about 1000 mg/kg, preferably fromabout 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2mg//kg to about 20 mg/kg, in one or more dose administrations daily, forone or more days, weekly, monthly, every two or three months, and soforth.

The microspheres may be administered alone or in combination with otherdrug therapies as part of a pharmaceutical composition. Such apharmaceutical composition may include the microspheres in combinationwith any standard physiologically and/or pharmaceutically acceptablecarriers which are known in the art. The compositions should be sterileand contain a therapeutically effective amount of the microspheres in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically-acceptable carrier” as used herein means one ormore compatible solid or liquid fillers, diluents or encapsulatingsubstances which are suitable for administration to a human or otheranimal. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application.

“Pharmaceutically acceptable” further means a non-toxic material that iscompatible with a biological system such as a cell, cell culture,tissue, or organism. The characteristics of the carrier will depend onthe route of administration. Physiologically and pharmaceuticallyacceptable carriers include diluents, fillers, salts, buffers,stabilizers, desiccants, bulling agents, propellants, acidifying agents,coating agents, solubilizers, and other materials which are well knownin the art. Carrier formulations suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated, and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing themicrospheres into association with a carrier which constitutes one ormore accessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the microspheres into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Additional examplesof solvents include propylene glycol, polyethylene glycol, vegetableoils such as olive oil, and injectable organic esters such as ethyloleate. Aqueous carriers include water, salts and buffer solutions suchas saline and buffered media, alcoholic/aqueous solutions and emulsionsor suspensions. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like. In general, the microspheres canbe administered to the subject (any mammalian recipient) using the samemodes of administration that currently are used for microparticletherapy in humans.

The microspheres are useful as therapeutic agents and may enable the useof alternative routes of administration when the microspheres include atherapeutic drug and are administered to a patient for slow release ortargeted delivery of the drug to the site requiring therapy. Themicrospheres are also useful as therapeutic or prophylactic agents whenthe microspheres include a macromolecule that is itself a therapeutic orprophylactic agent, such as an enzyme or immunoglobulin. The slowrelease of such therapeutic agents is particularly useful fortherapeutic proteins or peptides having short half-lives that must beadministered by injection.

The microspheres are useful for therapy or prophylaxis when themacromolecule is a therapeutic agent or a pharmaceutical compound thatis delivered to a patient and slowly released from the microspheres overtime. These microspheres are particularly useful for slow release ofdrugs with short biological half-lives, such as proteins or peptides. Ifthe pharmaceutical compound cannot be formed into a particle, then it iscompleted to a carrier, such as albumin, and the carrier-pharmaceuticalcompound complex is formed into a microsphere. The microsphere caneither provide for the slow release of the agent throughout the body orthe microsphere can include an affinity molecule specific for a targettissue, or tumor, and be injected into a patient for targeted slowrelease of the therapeutic agent, such as an antitumor, antiviral,antibacterial, antiparasitic, or antiarthritic agent, cytokine, hormone,or insulin directly to the site requiring therapy.

The following examples are illustrative of certain embodiments of theinvention, and are intended to further describe the present invention,without limiting it thereby. Various modifications can be made to theseembodiments without departing from the spirit or scope of the invention.

EXAMPLE 1 Preparation of Insulin Microspheres in Vessels of VariousSizes

Insulin microspheres are prepared according to the methods described inU.S. Pat. No. 5,981,719, the disclosure of which is incorporated hereinby reference in its entirety. The microspheres are formed byincorporating aqueous solutions of insulin, polyethylene glycol andpolyvinyl pyrrolidone in centrifuge tubes, and heating the solution totemperatures ranging from 37° C. up to 95° C. for a time period of about30 minutes.

Four (4) different size centrifuge tubes are used as follows: 1.5 mL, 15mL, 50 mL, and 100 mL. Microspheres of defined particle size andspherical shape are formed in the 1.5 mL tubes, but only amorphousprecipitates rather than microspheres are formed in the 15 mL, 50 mL and100 mL tubes. Attempts to form microspheres in the 15 mL, 50 mL and 100mL tubes using changes in heating conditions, pH, protein concentration,polymer concentration or type, heating time, or stirring conditionalteration are not successful, and microspheres cannot be reproduciblyformed in the larger tubes.

The surface area to volume ratio of the tubes is calculated. The 1.5 mLtube has a surface area to volume ratio of about 14 cm⁻¹; the 15 mL tubehas a surface area to volume ratio of about 6.5 cm⁻¹; and the 50 mL tubehas a surface area to volume ratio of about 3.5 cm⁻¹.

EXAMPLE 2 Preparation of Insulin Microspheres in Vessel Having a PackedBed

The production of insulin microspheres is undertaken in a containerhaving an increased surface area relative to the volume of theinsulin/polymer solution. In order to demonstrate the effects of arelatively high surface area, a polypropylene 50 mL centrifuge tube iscut into plastic shards approximately 5 cm in length and 1 cm in width.These plastic shards are then placed in a 15 mL polypropylene centrifugetube. 3.35 mL of a 10 mg/mL insulin solution in deionized water adjustedto pH 3 with 1 N HC1 is added to the centrifuge tube. 6.66 mL of 12.5%(wt/vol) polyethylene glycol (PEG, MW-3350 daltons), and 12.5% (wt/vol)polyvinyl pyrrolidone (PVP, MW-40,000) in 100 mM sodium acetate buffer,pH 5.6 is added to the 15 mL centrifuge tube. A control experiment isalso conducted using a similar 15 mL polypropylene tube containing nopolypropylene shards with the same quantity of insulin and PEG/PVPsolution as described above.

The solutions are heated in a 91° C. water bath without shaking for 15minutes. After this time, the tubes are centrifuged at 3200 rpm for 20minutes. The tubes are removed from the centrifuge, and the supernatantis discarded. Five (5) mL of deionized water is added to the pellet, andthe tube is vortexed to resuspend the pellet. The tube is thencentrifuged again at 3200 rpm for 20 minutes. This wash step isrepeated.

The microspheres are then subjected to particle size analysis by aCoulter laser light diffraction particle size analyzer.

The data in FIG. 1 shows that the particle size of greater than 95% ofthe insulin microspheres prepared according to the process of thisinvention is 1.5 microns by number average, surface area average andvolume average statistics. These close agreements in particle size areindicative of a very homogeneous microsphere population with no evidenceof aggregate formation.

Scanning electron micrographs (SEMs) of 1-2 micron insulin microspheresare prepared by lyophilizing the insulin microspheres, and sputtercoating them with gold. The microspheres are then observed under ascanning electron microscope.

An SEM of 1-2 micron insulin microspheres prepared according to theprocess of this invention is shown in FIG. 3, which shows evidence thatdiscrete, non-aggregated microspheres are formed. As a control, FIG. 2shows a sample of lyophilized insulin not subjected to the microspherefabrication conditions described herein. FIG. 2 shows a large amorphousmass of lyophilized insulin, with no evidence of microsphere formation.

A study conducted without the addition of plastic shards at the same 10mL scale yields aggregates seen in FIG. 4. FIG. 4 shows a mass ofaggregated material with very few microspheres present.

For tube volumes greater than 1.5 mL, particle size analysis indicatedthat without the presence of increased surface area effected by thepresence of the PP plastic shards, insulin microsphere aggregates andlarge particle formation were commonly observed. See FIG. 5.

FIG. 5 is a particle size analysis of microspheres prepared by a processoutside of the scope of the present invention, such as in large tubeswhich do not have their internal surface area increased by the presenceof polypropylene shards. FIG. 5 shows that the number average particlesize is 1.05 microns. This indicates that there are a lot of smallparticles. However, the surface area and volume average particle sizestatistics are 31.0 microns and 783.2 microns, respectively. Thisindicates that without adequate surface area in the fabrication tubes,large insulin aggregates also form. The particle size analysis in FIG. 5can be contrasted with FIG. 1 which shows no evidence of insulinaggregate formation.

EXAMPLE 3 Continuous Production of Protein Microspheres

An apparatus is assembled for the production of microspheres of uniformsmall size without aggregates. The apparatus contains a pump for pumpinginsulin/PEG/PVP solutions through narrow bore plastic tubing made frompolypropylene or other similar materials at elevated temperatures. Inthis manner, continuous flow-through methods can be used to continuouslyproduce microspheres in a controlled and reproducible fashion. The useof relatively small bore tubing ( 1/32 to ⅛ inch inner diameter),insures a high surface area to volume ratio, and submersing the tubingin a controlled temperature water bath permits the temperature to becontrolled until the formation of the microspheres is complete.

The following materials are used in this example:

-   Teflon (TFE 1/32″ inner diameter flexible tubing)-   Insulin (Calbiochem cat #40769)-   25% PEG/PVP pH 5.6 in 100 mM NaOAc buffer water bath at 90° C.    (American Scientific Products)

36.5 mg of insulin is weighed out and suspended in 3 mL of deionizedwater. 30 μL of 1 N HC1 is added to dissolve the insulin. The finalvolume of the solution is QS'ed to 3.65 mL with deionized water. 7.3 mLof PEG/PVP solution is then added to the insulin solution which is thenvortexed. This yields a homogeneous suspension of insulin and PEG/PVP.

The tubing is connected through a BioRad peristaltic pump running at aspeed of 0.4 mL/min. The tubing is submerged into the 90° C. water bath.The tubing exits the water bath and is inserted into a collection tubeimmersed in ice.

The flow rate is set at 0.4 mL/minute, and the total run time is 35minutes for the 10.95 mL volume. After collecting the microspheres, thecollection tube is spun at 3000 rpm for 20 minutes in a Beckman J613centrifuge. A 2nd water wash is completed, and the microsphere pelletsare spun down at 2600 rpm for 15 minutes. The final water is centrifugedat 1500 rpm for 15 minutes.

An aliquot is removed for particle size analysis by the Coulter LS 230.The microspheres are frozen at −80° C., and placed in a lyophilizer for2 days.

The particle size is determined to be 1.397 microns by volume, 1.119microns by surface area, and 0.691 microns by number. FIG. 6 is ascanning electron micrograph which indicates uniform sized andnon-aggregated insulin microspheres.

The use of the flow-through system, with the insulin exposed to 90° C.temperatures for a short time period, allows the production of particleswhich are 100% spherical microspheres. The final composition of themicrospheres is virtually all protein (insulin) as determined by HPLC.These results are consistent with the observation that plastic and evenglass shards that increase surface area to volume ratios result in theformation of microspheres rather than amorphous protein precipitates 70%of the starting material is incorporated into the insulin microspheres,as determined by the use of radio labeled insulin.

HPLC analysis of dissolved product indicates that the elution time ofdissolved insulin microspheres is not significantly different from aninsulin standard or the native insulin starting material.

EXAMPLE 4 Bioactivity of Insulin Microspheres

The bioactivity of insulin incorporated in the microspheres of thisinvention is demonstrated in the rat glucose depression model. Thepurpose of this experiment is to determine whether there is residualinsulin bioactivity in the insulin microspheres of this invention. Thisis accomplished by injecting animals-with both dissolved microspheres,and suspended microspheres prepared from Zn insulin.

The following materials are used in this experiment:

-   8 male Fisher rats with an average weight of 264 grams-   2×'s 2 mg of insulin microspheres deionized water-   0.5 cc insulin syringes-   AccuCheck Advantage glucose monitor (Roche Diagnostics)-   AccuCheck Comfort Curve glucose strips

The animals are divided into three groups: Group A, Group B and Group C.Each group contained three animals. The average weight of the animals inGroup A is 262 grams. The average weight of the animals in Group B is256 grams. The average weight of the animals in Group C is 280 grams.One mL of PBS is added to vial 1 and vortexed, and the particles aredissolved. One mL of deionized water is added to vial 2 and vortexed,and the particles remain in suspension. Each rat receives 200 μL ofinsulin (1 mg of insulin has 26 units of activity, and 1000 μL has 52units). Group A receives the PBS solubilized insulin particles. Group Breceives the suspended microspheres in deionized water. Group C receives200 μL of saline solution.

Blood glucose results are based on pre-bleeds and post-bleed injectionbleeds obtained by retro-orbital bleeding.

Glucose depression is demonstrated in both the insulin particle groupand the solubilized insulin particle group as shown in FIG. 7. Normalrats do not show significant deviations in blood glucose from thepre-injection values.

Based on the foregoing, bioactivity of insulin incorporated in ProMaxxmicrospheres is clearly demonstrated in the rat blood glucose depressionmodel. Blood glucose concentrations are depressed within 30 minutes ofinjection, and achieve their lowest levels 4 to 5 hours post injection.

Each of the foregoing patents, patent applications and references thatare recited in this application are herein incorporated in theirentirety by reference. Having described the presently preferredembodiments, and in accordance with the present invention, it isbelieved that other modifications, variations and changes will besuggested to those skilled in the art in view of the teachings set forthherein. It is, therefore, to be understood that all such variations,modifications, and changes are believed to fall within the scope of thepresent invention as defined by the appended claims.

1. A process for preparing microspheres comprising the steps ofcombining a macromolecule and a polymer in an aqueous solution, whereinthe polymer is water soluble or soluble in a water miscible solvent,contacting a volume of the aqueous solution with a surface at a surfacearea to volume ratio of at least about 6.5 cm⁻¹, heating the aqueoussolution, and forming the microspheres continuously in the aqueoussolution in a continuous process.
 2. The process of claim 1 wherein thesurface area to volume ratio is at least about 14 cm⁻¹.
 3. The processof claim 1 wherein the surface is a hydrophobic surface.
 4. The processof claim 3 wherein the hydrophobic surface is formed from a materialselected from metals, ceramics and glass.
 5. The process of claim 4wherein the metal is stainless steel.
 6. The process of claim 3 whereinthe hydrophobic surface is formed from a hydrophobic polymer.
 7. Theprocess of claim 6 wherein the hydrophobic polymer is selected from thegroup consisting of polypropylene, polystyrene, Teflon and siliconepolymers.
 8. The process of claim 1 wherein the macromolecule isselected from the group consisting of proteins, peptides, nucleic acids,carbohydrates, protein conjugates viruses, virus particles, and mixturesthereof.
 9. The process of claim 8 wherein the macromolecule is apeptide.
 10. The process of claim 9 wherein the peptide is apolypeptide.
 11. The process of claim 8 wherein the macromolecule is acarbohydrate.
 12. The process of claim 11 wherein the carbohydrate is apolysaccharide.
 13. The process of claim 8 wherein the macromolecule isa protein.
 14. The process of claim 13 wherein the protein is atherapeutic protein.
 15. The process of claim 14 wherein the protein isselected from the group consisting of insulin, human serum albumin,human growth hormone, parathyroid hormone and calcitonin.
 16. Theprocess of claim 1 wherein the microspheres have a mean diameter in therange of from about 0.1 microns to about 10.0 microns.
 17. The processof claim 16 wherein the microspheres have a mean diameter in the rangeof from about 0.5 microns to about 5.0 microns.
 18. The process of claim17 wherein the microspheres have a mean diameter in the range of fromabout 1.0 microns to about 2.0 microns.
 19. The process of claim 1wherein the aqueous solution of macromolecule and polymer is heated to atemperature in the range of from about 37° C. to about 95° C. for a timeperiod of about 1 minute to about 24 hours.
 20. The process of claim 1wherein the polymer is selected from the group consisting ofcarbohydrate polymers, polyaliphatic alcohols, poly(vinyl) polymers,polyacrylic acids, polyorganic acids, polyamino acids, polyethers,naturally occurring polymers, polyimides, polyesters, polyaldehydes,co-polymers, block co-polymers, terpolymers, surfactants, branchedpolymers, cyclo-polymers, and mixtures thereof.
 21. The process of claim20 wherein the polymer is selected from the group consisting of dextran,polyethylene glycol, polyvinyl pyrrolidone, co-polymers of polyethyleneglycol and polyvinyl pyrrolidone, polyvinyl alcohol, co-polymers ofpolyoxyethylene and polyoxypropylene, and mixtures thereof.
 22. Theprocess of claim 21 wherein the polymer is a co-polymer of polyethyleneglycol and polyvinyl pyrrolidone, or a co-polymer of polyoxyethylene andpolyoxypropylene.
 23. The process of claim 1 wherein the microspherescomprise greater than about 90% macromolecule by weight.
 24. The processof claim 23 wherein the microspheres comprise greater than about 95%macromolecule by weight.
 25. The process of claim 24 wherein themicrospheres comprise greater than about 99% macromolecule by weight.26. A product prepared according to the continuous process of claim 1.27. The process of claim 1 wherein the contacting of the aqueoussolution with the surface comprises moving the aqueous solutioncontinuously over the surface.
 28. The process of claim 27 wherein theaqueous solution is moved with a pump.
 29. The process of claim 27wherein the aqueous solution is moved at a speed of about 0.4 ml/minute.30. The process of claim 1 wherein the surface is the inner surface of atubing.
 31. The process of claim 30 wherein the tubing has an innerdiameter of 1/32 inches to ⅛ inches.
 32. The process of claim 1 furthercomprising combining additional polymers with the macromolecule and thepolymer in the aqueous solution.
 33. The process of claim 1 wherein theheating comprises exposing the solution to heat, radiation orionization.
 34. The process of claim 33 wherein the radiation ismicrowave radiation.
 35. The process of claim 33 wherein the heat,radiation or ionization is used in combination with sonication,vortexing, mixing or stifling.
 36. The process of claim 1, furthercomprising allowing the microspheres to continuously flow through forcollection.